Journal of the American College of Cardiology
2014 by the American College of Cardiology Foundation
Published by Elsevier Inc.
Vol. 63, No. 16, 2014
ISSN 0735-1097/$36.00
http://dx.doi.org/10.1016/j.jacc.2013.09.069
PRE-CLINICAL RESEARCH
Let-7g Improves Multiple Endothelial Functions
Through Targeting Transforming Growth
Factor-Beta and SIRT-1 Signaling
Yi-Chu Liao, MD, PHD,*y Yung-Song Wang, PHD,z Yuh-Cherng Guo, MD, MS,xk
Wen-Lien Lin, MS,z Ming-Hung Chang, MD,*y Suh-Hang Hank Juo, MD, PHDz{
Taichung, Taipei, and Kaohsiung, Taiwan
Objectives
The present study aimed to explore the role of microribonucleic acid (miRNA) Let-7g in regulating endothelial
functions.
Background
Derangement of miRNAs is implicated in the pathogenesis of cardiovascular diseases. Because the transforming
growth factor (TGF)-b pathway plays a regulatory role in endothelial functions, miRNAs targeted at TGF-b signal
cascade might affect vascular health.
Methods
Bioinformatics software predicted that Let-7g can influence the TGF-b pathway by targeting 3 genes. The Let-7g’s
effects on multiple endothelial functions were first tested in endothelial cells (ECs) and then in apolipoprotein
E knockout mice. Blood samples from lacunar stroke patients were also examined to further support Let-7g’s
effects on human subjects.
Results
Let-7g was experimentally confirmed to knock down the THBS1, TGFBR1, and SMAD2 genes in the TGF-b pathway.
PAI-I, one of the downstream effectors of the TGF-b pathway, was also down-regulated by Let-7g. Let-7g decreased
EC inflammation and monocyte adhesion and increased angiogenesis via the TGF-b pathway. Furthermore, Let-7g
reduced EC senescence through increasing SIRT-1 protein. Venous injection of Let-7g inhibitor into apolipoprotein
E knockout mice caused overgrowth of vascular intima-media, overexpression of PAI-1, increased macrophage
infiltration, and up-regulation of TGF-b downstream genes in the carotid arteries. Let-7g’s beneficial effects on EC
were reduced, whereas the TGF-b pathway was suppressed by ribonucleic acid interference. Restoration of the TGF-b
pathway also attenuated the effects of Let-7g overexpression. Low serum levels of Let-7g were associated with
increased circulating PAI-1 levels.
Conclusions
Decreased Let-7g levels impair endothelial function and increase the risks of cardiovascular diseases through
targeting TGF-b and SIRT-1 signaling. (J Am Coll Cardiol 2014;63:1685–94) ª 2014 by the American College of
Cardiology Foundation
The implications of microribonucleic acid (miRNA) in the
pathophysiology of cardiovascular diseases have recently
been recognized (1). Our group previously reported that
the human miRNA Let-7g prevents atherosclerosis by
inhibiting the uptake of oxidized low density lipoprotein
(ox-LDL) into endothelial cells (ECs) and vascular smooth
muscle cells (2). A recent study showed that patients with
coronary artery disease had decreased serum levels of
Let-7i (3), suggesting that the Let-7 family may play a
critical role in maintaining vascular health.
From the *Section of Neurology, Taichung Veterans General Hospital, Taichung,
Taiwan; yDepartment of Neurology, National Yang-Ming University School of Medicine,
Taipei, Taiwan; zDepartment of Genome Medicine, Kaohsiung Medical University,
Kaohsiung, Taiwan; xNeuroscience Laboratory, Department of Neurology, China
Medical University Hospital, Taichung, Taiwan; kSchool of Medicine, Medical College,
China Medical University, Taichung, Taiwan; and the {Department of Medical Research
and Department of Neurology, Kaohsiung Medical University Hospital, Kaohsiung,
Taiwan. This work was supported by funding from the National Science Council (Taiwan,
R.O.C. NSC 100-2923-B-037-001-MY3, 101-2314-B-075A-013, 101-2628-B-075A001-MY2, and 101-2628-B-037-003-MY2), National Health Research Institutes
(Taiwan, R.O.C. NHRI-Ex101-10107PI), Academia Sinica (BM102021169) Taichung
Veterans General Hospital (TCVGH-1013402C and TCVGH-1013404D) and
Kaohsiung Medical University Hospital grant (KMUH102-2T02). All authors have
reported that they have no relationships relevant to the contents of this paper to disclose.
Manuscript received June 5, 2013; revised manuscript received August 30, 2013,
accepted September 25, 2013.
See page 1695
The vascular endothelium is an active, dynamic tissue that is
indispensable for the regulation of vascular homeostasis
(4,5). The biological functions of healthy endothelium
1686
Liao et al.
Let-7g Improves Multiple Endothelial Functions
include: 1) promotion of vasodilation; 2) anticoagulation and
profibrinolysis effects; 3) inhibiADMA = asymmetric
tion of leukocyte adhesion and
dimethyl arginine
anti-inflammatory effects; and 4)
EC = endothelial cell
migration and angiogenesis (5).
HUVEC = human umbilical
Endothelial dysfunction precipendothelial cell
itates the atherosclerosis process
IL = interleukin
and is associated with an inMCP = monocyte
creased risk of coronary artery
chemotactic protein
disease, lacunar stroke, and ceremiRNA = microribonucleic
bral small vessel disease (6–8).
acid
The transforming growth facPAI = plasminogen activator
tor
(TGF)-b pathway has been
inhibitor
recognized
as playing a regulatory
qPCR = quantitative
role
in
endothelial
functions
real-time polymerase chain
reaction
(9,10). The specific aim of the
present study was to test whether
SIRT = sirtuin
Let-7g can regulate the TGF-b
SMAD2 = SMAD family
member 2
pathway and influence other
mechanisms to affect endothelial
TGF = transforming growth
factor
functions. First, the TargetScan
algorithm, which uses primarily
THBS1 = thrombospondin-1
sequence complementarity beVCAM = vascular cell
adhesion molecule
tween messenger ribonucleic
acids (RNAs) and the seed region
of miRNA (11), was used to search for Let-7g target genes
involved in the TGF-b signaling pathway. Experiments
were conducted to confirm the 3 predicted target genes
and to test for the consequences of EC functions after
inhibiting or overexpressing Let-7g. Plasminogen activator
inhibitor (PAI)-1, asymmetric dimethyl arginine (ADMA),
vascular cell adhesion molecule (VCAM)-1, monocyte
chemotactic protein (MCP)-1, and interleukin-6 (IL-6)
were used to serve as EC biomarkers (12–14). RNA interference experiments were used to block TGF-b/SMAD
signaling to further confirm that Let-7g’s beneficial effects
on ECs were primarily mediated by this signaling. In
addition, the relationship between EC senescence and
Let-7g was explored. The present study used experimental
animals and human samples to verify the impact of Let-7g
in vivo.
Abbreviations
and Acronyms
Methods
Please see the Online Appendix for detailed methods.
Construction of the 30 -untranslated region reporter
plasmids. Briefly, the plasmid-containing predicted Let-7g
binding site of each gene was reacted with Let-7g mimic or
Let-7g inhibitor to confirm that Let-7g can directly bind to
the target 30 -untranslated region (UTR).
miRNA transfection. Human umbilical endothelial cells
(HUVECs) were transfected with the following miRNAs
(5 nM): mimic control, inhibitor control, Let-7g mimic
(MC11758), and Let-7g inhibitor (MH11758) using
HiPerFect transfection reagent (Qiagen, Valencia, California).
JACC Vol. 63, No. 16, 2014
April 29, 2014:1685–94
Two additional approaches were used to further support
the fact that Let-7g’s effects on ECs are mediated through
the TGF-b pathway: 1) restoration of the pathway by
treating HUVECs with TGF-b1 (10 ng/ml, Sigma-Aldrich,
St. Louis, Missouri) after transfection of Let-7g mimic
for 24 h; and 2) suppression of TGF-b/SMAD2 signaling
by transfecting SMAD2 small interfering RNA (siRNA)
and/or TGFBR1 siRNA while transfecting Let-7g inhibitor.
Assays for endothelial functions. Detailed methods used
for monocyte adhesion assay; tubule formation to assess
angiogenesis; measurements of VCAM-1, MCP-1, IL-6,
and PAI-1 concentrations; assay to estimate endothelial
nitric oxide synthase (eNOS) activity; and the b-galactosidase
(b-gal) assay for senescent status of HUVECs can be found in
the Online Appendix.
Immunofluorescence stain. See the Online Appendix.
Animal model. Lentiviral expression vector pCDH-CMVMCS-EF1-GreenPuro (SBI Mountain View, California)
was used to carry Let-7g–expressing plasmids or Let-7g
sponge plasmids. The inserts of Let-7g overexpressing plasmids and sponge plasmids were synthetic oligonucleotides
containing EcoRI and XhoI overhangs. The empty vector
was used as a negative control. Apolipoprotein E-knockout
(ApoE-KO) mice were fed 4 g/day of high-fat diet (product
code 57BD, Testdiet, St. Louis, Missouri) to elicit fatty streak
lesions in the arteries. Lentivirus carrying different types of
plasmids was used: empty or Let-7g–expressing plasmids for
12-week treatment and empty or Let-7g sponge plasmids
for 9-week treatment. Undiluted viral stocks (200 to 250 ml)
were injected into the tail veins of ApoE-KO mice (n ¼ 2 for
each group) weekly in the indicated time periods.
Stains for the carotid artery. Briefly, the slices of carotid
arteries were stained with anti–PAI-1 antibody (1:100
dilution) or anti-pSMAD2 antibody (1:2,000 dilution), and
then the stain intensity was quantified (see details in the
Online Appendix). To stain macrophages, the slices were
incubated with anti–Mac-3 antibody (1:100 dilution;
Millipore Billerica, Massachusetts) and Dylight 649 conjugated goat antirat antibody (1:200 dilution; Rockland Gilbertsville, Pennsylvania).
Human study. Sixty patients with lacunar infarction were
enrolled from the Taichung Veterans General Hospital in
Taiwan. All participants provided written informed consent,
and the study protocols were approved by the local Institutional Review Board (VGHTC-C10253). The patients’
serum levels of Let-7g and RNA-U6B were determined by
quantitative real-time polymerase chain reaction (qPCR).
Plasma PAI-1 and ADMA levels were determined by
enzyme-linked immunosorbent assay for patients with the
lowest or highest tertile of Let-7g levels (n ¼ 20 per group).
Five persons were excluded because samples were contaminated by hemolysis and severe hyperlipidemia.
Statistical analysis. For cellular studies, variables are presented as mean SE from 3 independent experiments.
Student t test was used to compare the variables between the
treatment and control groups and the variables between
JACC Vol. 63, No. 16, 2014
April 29, 2014:1685–94
stroke patients with the lowest and highest tertile of Let-7g.
The data from each experiment in the cellular study can be
found in the Online Appendix. Three replicates were performed in each cellular experiment, and the number of
replicates was used as the unit of analysis. For human blood
samples, variables are presented as mean SD. A 2-sided
p value <0.05 was considered significant. Owing to the
small sample size in cellular experiments and human studies,
we did not conduct the Bonferroni correction for multiple
comparisons or test for normality and homogeneity.
Results
Confirmation of the Let-7g target genes THBS1,
TGFBR1, and SMAD2. Let-7g binding sites were predicted at the 30 -UTRs of the thrombospondin 1 (THBS1),
transforming growth factor beta receptor 1 (TGFBR1), and
SMAD family member 2 (SMAD2) genes (Fig. 1A).
Plasmids carrying either the wild-type or mutant sequence
of 30 -UTR of each gene were constructed (Fig. 1A, Online
Table 1) and transfected to HEK293 cells. For the wild-type
Figure 1
Liao et al.
Let-7g Improves Multiple Endothelial Functions
1687
sequence of THBS1, Let-7g mimic induced a dosedependent decrease of luciferase activity, whereas Let-7g
inhibitor exerted an opposite effect (Figs. 1B and 1C, left
panel). For the mutant sequence, Let-7g mimic had only a
mild knock-down effect, and Let-7g inhibitor paradoxically
decreased luciferase activity (Figs. 1B and 1C, left panel).
Similarly, Let-7g mimic induced a stepwise decrease of
luciferase activity in plasmids carrying the wild-type 30 -UTR
sequence of TGFBR1 or SMAD2 but had no effect on
plasmids carrying the mutant sequences (Fig. 1B, middle
and right panels). On the contrary, Let-7g inhibitor caused a
dose-dependent increase in luciferase activity for plasmids
carrying the wild-type 30 -UTR sequences but did not have
such an effect on plasmids carrying the mutant sequences
(Fig. 1C, middle and right panels). The results from the
pMIR-reporter assay provided direct evidence of Let-7g0 s
binding to the 3 target genes.
Let-7g affected expression levels of the 3 target genes.
qPCR confirmed successful transfection of Let-7g mimic or
inhibitor to alter the intracellular Let-7g levels (Online
Fig. 1). As shown in Figure 2A, Let-7g mimic reduced
Confirmation of the Let-7g Target Sites at the 30 -UTRs of THBS1, TGFBR1, and SMAD2
(A) Wild-type sequences (top) and mutant sequences (bottom) at the Let-7g target sites. Effects of Let-7g mimic (B) and Let-7g inhibitor (C) on luciferase activity. HEK293 cells
were transfected with 700 ng of pMIR-reporter plasmids with wild-type (open bars) or mutant (solid bars) sequence and different doses of Let-7g mimic or inhibitor for 24 h.
*p < 0.05; **p < 0.01. TGFBR ¼ transforming growth factor beta receptor; THBS1 ¼ thrombospondin-1; UTR ¼ untranslated region.
1688
Figure 2
Liao et al.
Let-7g Improves Multiple Endothelial Functions
JACC Vol. 63, No. 16, 2014
April 29, 2014:1685–94
Let-7g Influenced the Expression Levels of the 3 Target Genes in HUVECs and Affected the Phosphorylation of SMAD2
(A to C) Relative messenger ribonucleic acid levels are shown. (D) Western blots are shown of total SMAD2 protein and (E) phosphorylated SMAD2 (pSMAD2). The pSMAD2to-total SMAD2 ratio was used to estimate SMAD2 activity (right). *p < 0.05; **p < 0.01. (F) Immunofluorescence staining for SMAD2 (green) and 4’,6-diamidino-2phenylindole (blue) in HUVECs. Original magnification 600. Bar ¼ 20 mm. ctrl ¼ control; GADPH ¼ glyceraldehyde 3-phosphate dehydrogenase; HUVECs ¼ human umbilical
endothelial cells; other abbreviations as in Figure 1.
JACC Vol. 63, No. 16, 2014
April 29, 2014:1685–94
THBS1 expression, whereas Let-7g inhibitor increased
THBS1 expression levels (p < 0.01). Similarly, Let-7g
mimic knocked down TGFBR1 and SMAD2 expression
levels, whereas Let-7g inhibitor increased their transcription
(p < 0.05) (Figs. 2B and 2C). In addition, Western blots
illustrated the fact that Let-7g mimic significantly reduced
the protein amount of total SMAD2 (p < 0.05) (Fig. 2D).
Given that TGFBR1 and SMAD2 behave as check points in
the TGF-b signal cascade (15), Let-7g might interfere with
the signal pathway by also influencing SMAD2 phosphorylation. To test this hypothesis, HUVECs were treated
with TGF-b1, which caused a rapid but transient increase
in phosphorylated SMAD2 (pSMAD2). Let-7g mimic
diminished TGF-b1–induced pSMAD2, whereas Let-7g
inhibitor significantly increased pSMAD2 under TGF-b1
stimulation (Fig. 2E, left panel). The pSMAD2-to-total
SMAD2 ratio was used to indicate the changes in SMAD2
activity. Let-7g mimic influenced both total and pSMAD2
but did not significantly affect the pSMAD2/SMAD2 ratio
(Fig. 2E, middle panel). However, Let-7g inhibitor significantly increased the ratio (Fig. 2E, right panel), which implied
that loss-of-function in Let-7g can be more influential than
gain-of-function in regulating the TGF-b pathway.
The immunofluorescence stain also supported the fact
that Let-7g had dual effects on SMAD2. HUVECs transfected with Let-7g mimic had less SMAD2 in the nucleus
(i.e., less pSMAD2) as indicated by a weaker stain in the
nucleus (Fig. 2F, left panel). In contrast, Let-7g inhibitor
increased SMAD2 levels in the nucleus and cytoplasm
(Fig. 2F, right panel).
Let-7g inhibited cell adhesion and inflammation but
increased angiogenesis. Because the TGF-b pathway has
pleiotropic effects on vascular pathophysiology (9,10), we
further tested the effect of Let-7g on EC functions,
including monocyte adhesion, inflammation, and angiogenesis. Our results showed that Let-7g mimic significantly
reduced VCAM-1 secretion (Fig. 3A), which subsequently
inhibited the monocyte adhesion to HUVECs (Fig. 3B).
Monocyte adhesion was reduced when intracellular Let-7g
levels were increased by Let-7g mimic (p < 0.001)
(Fig. 3B, middle panel). In contrast, monocyte adhesion was
aggravated when HUVECs were transfected with Let-7g
inhibitor (Fig. 3B, left panel). Activating the TGF-b
pathway by TGF-b1 reversed the inhibitory effect of Let-7g
mimic on monocyte adhesion, whereas blocking the signal
cascade by SMAD2/TGFBR1 siRNAs diminished the
detrimental effects of Let-7g inhibitor (Fig. 3B).
The secretion of inflammatory cytokines (MCP-1 and
IL-6) was significantly reduced in HUVECs transfected
with Let-7g mimic (p < 0.05) (Fig. 3C). Similarly, Let-7g’s
anti-inflammatory effect was reversed after restoration of the
TGF-b signaling (Online Fig. 2). The proinflammatory
effects of Let-7g inhibitor were reduced when the TGF-b
signal was blocked (Online Fig. 3). Let-7g mimic mildly
increased angiogenesis by the matrigel assay (BD biosciences, Franklin Lakes, New Jersey) (Fig. 3D, middle
Liao et al.
Let-7g Improves Multiple Endothelial Functions
1689
panel), and treatment with TGF-b1 (10 ng/ml) did not
significantly reverse Let-7g’s effect (Fig. 3D, right panel). In
contrast, the tubule formation was significantly inhibited in
HUVECs transfected with Let-7g inhibitor (p ¼ 0.005).
Suppression of the TGF-b signaling by siRNAs recovered
the angiogenic ability (Fig. 3D, right panel)
Let-7g inhibited in vitro and in vivo PAI-1
expression. PAI-1 levels were used to imply the impact
of Let-7g on TGF-b–regulated cardiovascular risks. In
HUVECs, Let-7g mimic mildly inhibited PAI-1 expression,
whereas Let-7g inhibitor significantly increased PAI-1
messenger RNA levels (Fig. 4A). The secretion of PAI-1
was significantly reduced in HUVECs transfected with
Let-7g mimic compared with those transfected with the
mimic control (p < 0.05) (Fig. 4B).
We then detected the amounts of PAI-1 and pSMAD2
protein in the carotid arteries of ApoE-KO mice fed a highfat diet for 9 or 12 weeks (Fig. 4C). Lentivirus carrying Let-7g
expressing plasmids was injected into the tail veins of mice
each week to increase endogenous Let-7g levels, whereas
lentivirus carrying Let-7g sponge plasmids reduced Let-7g
levels. The treatment of Let-7g–expressing plasmids
increased arterial Let-7g levels by 2.6-fold, whereas Let-7g
sponge plasmids reduced arterial Let-7g levels to 48%.
The immunohistochemistry stain showed reduced amounts
of PAI-1 and pSMAD2 protein in mice treated with
Let-7g–expressing plasmids (Fig. 4C, left panel). On the
contrary, Let-7g sponge plasmids caused overgrowth of the
intima-media layer and overexpression of PAI-1 and
pSMAD2 in the vessel walls. Quantitative analysis of the
PAI-1 and pSMAD2 staining further demonstrated that
loss-of-function in Let-7g had a stronger impact than gainof-function (Fig. 4C, right panel). The qPCR data consistently supported the findings from immunohistochemistry
stain in that PAI-1 level was decreased to 28% (p < 0.001)
and SMAD2 level was decreased to 44% (p < 0.001) in the
Let-7g–treated mice at 12 weeks (Online Fig. 4). For the
mice treated with Let-7g sponge, PAI-1 and SMAD2 levels
were increased by 2.6-fold (p ¼ 0.02) and 1.9-fold (p ¼
0.003) at 9 weeks, respectively (Online Fig. 4).
Let-7g inhibited monocyte infiltration and TGF-b
downstream genes in vivo. In vivo, monocyte infiltration
can be measured by the number of macrophages in the
carotid arteries of ApoE-KO mice. Figure 4D shows that
the overexpressed Let-7g could reduce macrophage infiltration and vice versa. The messenger RNA levels of
THBS1, TGFBR1, VCAM-1, MCP-1, and IL-6 in carotid
arteries were in concert with the findings in cellular experiments (Fig. 4E). These TGF-b downstream genes were
suppressed in mice with Let-7g overexpression (p < 0.01)
(Fig. 4E) and were up-regulated in mice treated with Let-7g
sponge (p < 0.05) (Fig. 4E). Our findings provided in vivo
support for the Let-7g effects on vascular morphology and
downstream effectors of TGF-b signaling pathway.
Let-7g and lacunar stroke. Endothelial dysfunction is one
of the pathogeneses underlying lacunar stroke (7,8). To
1690
Figure 3
Liao et al.
Let-7g Improves Multiple Endothelial Functions
JACC Vol. 63, No. 16, 2014
April 29, 2014:1685–94
Let-7g Overexpression Inhibited Cell Adhesion and Secretion of Inflammatory Cytokines But Increased Angiogenesis
Concentrations of (A) vascular cell adhesion molecule (VCAM)-1, (C) monocyte chemotactic protein (MCP)-1, and interleukin (IL)-6 are shown. (B) Adhesion of THP-1 cells
to HUVECs and (D) angiogenesis of HUVECs were assessed by monocyte adhesion assay and matrigel assay, respectively. *p < 0.05; **p < 0.01. Abbreviations as
in Figures 1 and 2.
JACC Vol. 63, No. 16, 2014
April 29, 2014:1685–94
Figure 4
Liao et al.
Let-7g Improves Multiple Endothelial Functions
1691
Let-7g Inhibited PAI-1 Expression, Macrophage Adhesion, and TGF-b Signal Pathway In Vivo
(A) Relative messenger ribonucleic levels and (B) concentrations of PAI-1 are shown. (C) Representative slices of carotid artery stained with PAI-1 (brown, top) and pSMAD2
(brown, bottom) from apolipoprotein E-knockout mice. Overgrowth of intima-media layer in carotid arteries is indicated by an arrow. Bar ¼ 50 mm. (D) Immunofluorescence
stains indicate macrophage in the carotid artery. (E) Expression levels of downstream genes in the transforming growth factor (TGF)-b signal pathway are shown. HF ¼ high-fat
diet containing 0.15% cholesterol; other abbreviations as in Figures 1 and 2.
1692
Liao et al.
Let-7g Improves Multiple Endothelial Functions
determine whether Let-7g could modulate EC functions in
human subjects, we first measured the serum Let-7g levels
in 60 patients with lacunar infarction who were likely to
have EC dysfunction. These patients, whose Let-7g levels
were in the lowest or highest tertile, were selected for
measurement of their plasma levels of PAI-1 and ADMA,
which are biomarkers of endothelial dysfunction (13,14).
The data showed that plasma PAI-1 levels were significantly higher in the patients with low Let-7g levels than in
patients with high Let-7g levels (p ¼ 0.04) (Online
Table 2). However, there were no differences in plasma
levels of ADMA between the patients with high and low
Let-7g levels.
Let-7g prevented endothelial senescence through
influencing SIRT-1. We further tested whether Let-7g
could alleviate endothelial senescence. Sirtuin 1 (SIRT-1)
was selected as a senescence marker, although it was not
predicted as a Let-7g target gene. Transfection of Let-7g
mimic did not change the messenger RNA levels of SIRT-1
in HUVECs (data not shown). However, the SIRT-1
protein levels were significantly increased in HUVECs
transfected with Let-7g mimic (Fig. 5A). To determine
whether Let-7g prevented EC senescence through increasing
SIRT-1 expression, nicotinamide was added to the culture
medium to inhibit endogenous SIRT-1 activity (Fig. 5B).
Compared to the control group, Let-7g mimic significantly
reduced the percentage of senescent cells. As expected,
Let-7g inhibitor caused the opposite effect by increasing
the percentage of senescent cells. Although SIRT-1 could
Figure 5
JACC Vol. 63, No. 16, 2014
April 29, 2014:1685–94
increase eNOS activity (16), we did not find any differences
between eNOS activity in HUVECs transfected with mimic
control and that in Let-7g mimic (Fig. 5C).
Discussion
The present study demonstrated that Let-7g can improve
several endothelial functions including decrease of senescence, inflammation, and monocyte adhesion, and increase in
angiogenesis. The beneficial effects of Let-7g may be
mediated by several regulatory pathways, but the present
study focused primarily on TGF-b and SIRT-1 signaling
(Fig. 6). First, we proved that Let-7g directly regulated 3
target genes (THBS1, TGFBR1, and SMAD2) in TGF-b
signaling. Second, Let-7g was shown to indirectly increase
SIRT-1 expression to prevent EC senescence. Third, the
interventional study using ApoE-KO mice and the observational study using clinical human samples further verified
Let-7g’s beneficial effects on endothelial functions. Although
miRNAs have been extensively investigated in cancers, their
roles and mechanisms in the cardiovascular system are still
underexplored. Let-7 family has been heavily studied in
different cancers (17), but its role in vascular biology remains
unclear. The strengths/merits of the present study are to
combine an array of genome and miRNA techniques along
with immunohistochemical staining and microscopy images.
We used both gain- and loss-of-function of Let-7g to validate its important roles in EC functions. Moreover, RNA
interference experiments were conducted to block TGF-b/
Let-7g Prevented Endothelial Senescence Through SIRT-1 Up-Regulation
(A) Western blot shows SIRT-1 protein. (B) b-galactosidase (b-Gal) staining of HUVECs to quantify the percentage of senescent cells. (C) Endothelial nitric oxide synthase
(eNOS) activity was measured using the Griess method. *p < 0.05; **p < 0.01. Abbreviations as in Figures 1 and 2.
JACC Vol. 63, No. 16, 2014
April 29, 2014:1685–94
Figure 6
Schematic Shows Pleotropic Effects of Let-7g
on Endothelial Functions
Abbreviations as in Figures 1 and 3.
SMAD signaling to further confirm that Let-7g’s beneficial
effects on EC were primarily mediated by the TGF-b
signaling. The consistent results from cellular experiments,
animal models, and human data supported Let-7g as an
important regulator to maintain the healthy vascular
endothelium.
The present study demonstrated that Let-7g’s functions
in keeping endothelial homeostasis were mediated primarily
through the TGF-b pathway. THBS1 functions as a major
activator of TGF-b pathway by converting the latent
TGF-b procytokine to its biologically active form (18).
THBS1-activated TGF-b pathway can increase the
expression of adhesion molecules and promote monocyte
adhesion to ECs (19). Our data showed that knock down of
THBS1 as well as 2 downstream genes (TGFBR1 and
SMAD2) by Let-7g led to a decrease of monocyte adhesion
in vivo and in vitro. THBS1 is a potent inhibitor of
angiogenesis (20). Indeed, we showed that enhancing the
signal transduction of the THBS1/TGFBR1/SMAD2
pathway by Let-7g inhibitor disturbed the EC activation
during angiogenesis.
Our animal studies found that a decrease of arterial Let-7g
levels resulted in up-regulation of pSMAD2 and PAI-1 and
accelerated intima-media overgrowth in the carotid arteries.
These findings were in line with reports of increased neointimal formation in the carotid arteries by overexpressed
TGF-b1 (21). A recent study reported that pSMAD2 was
expressed mainly in ECs covering atherosclerotic plaques
(22), which is consistent with our findings from animal
models (Online Fig. 5).
It should be noted that our results indicated that lossof-function in Let-7g can be more influential than gainof-function in regulating TGF-b pathway and cell
senescence. Given that the Let-7 family plays critical roles
Liao et al.
Let-7g Improves Multiple Endothelial Functions
1693
in cell differentiation (23), normal cells, including ECs,
have high levels of endogenous Let-7. Therefore, the
supply of exogenous Let-7g may not affect cellular functions as substantially as the supply of Let-7g inhibitor.
These observations suggested that more attention should
be paid to a human subject with decreased Let-7g levels.
Lacunar stroke accounts for one-fourth of all ischemic
stroke events. Lacunar stroke is caused by occlusion of the
small and deep perforating (lenticulostriate) arteries. The
pathogenic changes in these types of arteries have been
referred to as “lipohyalinosis” or “fibrinoid necrosis” (24), but
the exact initiating cause of these vascular abnormalities
remains unknown. Endothelial dysfunction can be an underlying cause of lacunar stroke (7,8). Among our lacunar
stroke patients, higher PAI-1 levels were associated with
lower serum levels of Let-7g. Elevated plasma levels of
PAI-1 have been documented in patients with acute
ischemic stroke (14). The present study elucidated how
Let-7g can regulate multiple EC functions; therefore,
decreased levels of Let-7g may be a risk factor for lacunar
infarction due to its involvement in endothelial functions.
Endothelial dysfunction can be attributed partially to EC
senescence. SIRT-1 has been implicated in antiaging and
anti-inflammation (25) and also may regulate EC functions
through other downstream targets (26). Although studies
have implied Let-7’s effect on antiaging process (27), our
finding of up-regulation of SIRT-1 by Let-7g has not been
reported before. It is likely that Let-7g is an indirect
regulator for SIRT-1 because there is no Let-7g binding
site at the 30 -UTR of SIRT-1. The MetaCore network
prediction identified several possible pathways to account
for Let-7g’s effect on SIRT-1 (Online Fig. 6, Online
Appendix). For example, Let-7g could inhibit Fas, which
activates the death domain association protein (DAXX).
DAXX is a negative regulator of tumor protein p53, which
increases SIRT-1 expression via 2 validated binding sites
at the SIRT-1 promoter (28). Further studies are warranted
to depict Let-7g relationship with the complex SIRT-1
signaling.
Study limitations. First, we did not test for the potential
pathways linking let-7g and SIRT-1. Second, we cannot
deduce let-7g’s effect toward all stroke subtypes since only
patients with lacunar infarction were included in the present
study. Third, let-7a, let-7b, and let-7c also have been related
to stroke; therefore, other members of let-7g family could
also affect EC functions.
Conclusions
Our cellular, animal, and human studies confirmed that the
regulation of TGF-b signaling and SIRT-1 expression by
Let-7g leads to beneficial effects on EC. The present study
provides new evidence of the involvement of Let-7g in
multiple endothelial functions. Given that healthy ECs are
crucial to maintain normal functions of small arterioles
and to prevent atherosclerosis in large and medium-sized
1694
Liao et al.
Let-7g Improves Multiple Endothelial Functions
arteries, maintenance of a sufficient Let-7g level may reduce
the risk of cardiovascular diseases.
Reprint requests and correspondence: Dr. Suh-Hang H. Juo,
Department of Genome Medicine, Kaohsiung Medical University,
100 TzYou First Road, Kaohsiung 80708, Taiwan. E-mail: hjuo@
kmu.edu.tw.
REFERENCES
1. Ono K, Kuwabara Y, Han J. MicroRNAs and cardiovascular diseases.
FEBS J 2011;278:1619–33.
2. Chen KC, Hsieh IC, Hsi E, et al. Negative feedback regulation between
microRNA Let-7g and the oxLDL receptor LOX-1. J Cell Sci 2011;
124:4115–24.
3. Satoh M, Tabuchi T, Minami Y, Takahashi Y, Itoh T, Nakamura M.
Expression of Let-7i is associated with Toll-like receptor 4 signal in
coronary artery disease: effect of statins on Let-7i and Toll-like receptor
4 signal. Immunobiology 2012;217:533–9.
4. Gibbons GH. Endothelial function as a determinant of vascular function and structure: a new therapeutic target. Am J Cardiol 1997;79:3–8.
5. Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction: a marker
of atherosclerotic risk. Arterioscler Thromb Vasc Biol 2003;23:168–75.
6. Vanhoutte PM. Endothelial dysfunction: the first step toward coronary
arteriosclerosis. Circ J 2009;73:595–601.
7. Hassan A, Hunt BJ, O’Sullivan M, et al. Markers of endothelial
dysfunction in lacunar infarction and ischaemic leukoaraiosis. Brain
2003;126:424–32.
8. Knottnerus IL, Ten Cate H, Lodder J, Kessels F, van Oostenbrugge RJ.
Endothelial dysfunction in lacunar stroke: a systematic review. Cerebrovasc Dis 2009;27:519–26.
9. Bobik A. Transforming growth factor-betas and vascular disorders.
Arterioscler Thromb Vasc Biol 2006;26:1712–20.
10. Goumans MJ, Liu Z, ten Dijke P. TGF-beta signaling in vascular
biology and dysfunction. Cell Res 2009;19:116–27.
11. Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked
by adenosines, indicates that thousands of human genes are microRNA
targets. Cell 2005;120:15–20.
12. Constans J, Conri C. Circulating markers of endothelial function in
cardiovascular disease. Clin Chim Acta 2006;368:33–47.
13. Badimon L, Romero JC, Cubedo J, Borrell-Pages M. Circulating
biomarkers. Thromb Res 2012;130 Suppl 1:S12–5.
14. Tjarnlund-Wolf A, Brogren H, Lo EH, Wang X. Plasminogen activator inhibitor-1 and thrombotic cerebrovascular diseases. Stroke 2012;
43:2833–9.
JACC Vol. 63, No. 16, 2014
April 29, 2014:1685–94
15. Schmierer B, Hill CS. TGFbeta-SMAD signal transduction: molecular
specificity and functional flexibility. Nat Rev Mol Cell Biol 2007;8:
970–82.
16. Mattagajasingh I, Kim CS, Naqvi A, et al. SIRT1 promotes
endothelium-dependent vascular relaxation by activating endothelial
nitric oxide synthase. Proc Natl Acad Sci U S A 2007;104:14855–60.
17. Boyerinas B, Park SM, Hau A, Murmann AE, Peter ME. The role of Let7 in cell differentiation and cancer. Endocr Relat Cancer 2010;17:F19–36.
18. Murphy-Ullrich JE, Poczatek M. Activation of latent TGF-beta by
thrombospondin-1: mechanisms and physiology. Cytokine Growth
Factor Rev 2000;11:59–69.
19. Narizhneva NV, Razorenova OV, Podrez EA, et al. Thrombospondin-1
up-regulates expression of cell adhesion molecules and promotes
monocyte binding to endothelium. FASEB J 2005;19:1158–60.
20. Lawler J. Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth. J Cell Mol Med 2002;6:1–12.
21. Otsuka G, Agah R, Frutkin AD, Wight TN, Dichek DA. Transforming growth factor beta 1 induces neointima formation through
plasminogen activator inhibitor-1-dependent pathways. Arterioscler
Thromb Vasc Biol 2006;26:737–43.
22. Vecerova L, Strasky Z, Rathouska J, et al. Activation of TGF-beta
receptors and Smad proteins by atorvastatin is related to reduced
atherogenesis in ApoE/LDLR double knockout mice. J Atheroscler
Thromb 2012;19:115–26.
23. Roush S, Slack FJ. The Let-7 family of microRNAs. Trends Cell Biol
2008;18:505–16.
24. Fisher CM. Lacunar strokes and infarcts: a review. Neurology 1982;32:
871–6.
25. Stein S, Matter CM. Protective roles of SIRT1 in atherosclerosis. Cell
Cycle 2011;10:640–7.
26. Yu W, Fu YC, Chen CJ, Wang X, Wang W. SIRT1: a novel target to
prevent atherosclerosis. J Cell Biochem 2009;108:10–3.
27. Benhamed M, Herbig U, Ye T, Dejean A, Bischof O. Senescence is an
endogenous trigger for microRNA-directed transcriptional gene
silencing in human cells. Nat Cell Biol 2012;14:266–75.
28. Nemoto S, Fergusson MM, Finkel T. Nutrient availability regulates
SIRT1 through a forkhead-dependent pathway. Science 2004;306:
2105–8.
Key Words: endothelial function
pathway.
-
Let-7g
-
PAI-1
-
SIRT-1
-
TGF-b
APPENDIX
For an expanded Materials and Methods section as well as supplemental
tables and figures, please see the online version of this article.